PROJECT ABSTRACT: The overall goal of this grant is to develop improved methods of treating axial back pain via extracorporeal application of non-thermal or mechanical pulsed focused ultrasound (p-FUS) Recent in vitro studies have shown that low intensity pulsed ultrasound (LIPUS) stimulation can induce disease-modifying activities in intervertebral disc cells. These observations suggest that LIPUS or p-FUS may be a valuable non-invasive therapeutic strategy for the estimated 3 million low back pain sufferers who have failed conservative therapy and whose only other option is risky, spinal surgery. However, practicable p-FUS dose settings that optimize desirable disc cell behaviors and tissue regeneration have not been established; further, methods to deliver ultrasound energy to the disc appear feasible based upon ultrasound imaging protocols applied to the spine, but practical methods to deliver the energy for LIPUS to the disc remain to be evaluated. The goals of this R21 application are to: 1) use an established 3D human degenerative disc cell culture system to characterize and optimize cell behaviors in response to varying LIPUS or p-FUS regimens, using a custom calibrated ultrasound exposimetry system specifically designed to deliver a targeted ultrasound intensity while preventing heating, reflections, and standing waves within cell culture plates; 2) establish the LIPUS disease-modifying activity in vivo using an established rat-tail injury model, using a p-FUS system designed specifically for isolated calibrated exposure of the targeted disc; and 3) apply existing diagnostic data sets and employ complex 3D acoustic and thermal modeling to define feasibility and limitations of clinical p- FUS delivery. Points of innovation include a novel approach for in vitro studies to deliver quantifiable LIPUS without sample heating or standing waves, a broad optimization of frequency and energy effects of LIPUS on damaged disc cells as required for consideration of deep targets in the spine, and to also investigate and characterize components of the healing and remodeling response in vivo. This research team has significant experience with developing, characterizing, and the in vivo evaluation of ultrasound systems as well as in vitro and in vivo models of intervertebral disc degeneration and regeneration. Consequently, these proposed studies with important clinical implications and significance have high probability of being completed successfully. Completion of this proposed study has potential to lead to a novel and improved non-invasive method for treating back injury and related pain.